The main objective of this proposal is the development of automated, high-throughput, Caenorhabditis elegans-based assays that can be used to screen chemical compounds and identify those with antifungal activity. This whole animal approach provides an unambiguous assay endpoint in the survival/death of the worms, allows the use of liquid handling robots for filling assay plates and for pin transfer of compounds from library stock plates to assay plates, and permits automated readouts using plate readers and imaging microscopes. In preliminary studies, we found that key components of Candida albicans and Cryptococcus neoformans pathogenesis in mammals are also involved in nematode killing. We used these observations to devise whole-animal C. elegans-C. albicans and C. elegans-C. neoformans assays that are performed using 96-well plate technology and study of fungal cells that are in non-planktonic form and identification of antifungal compounds in a system where both the pathogen and the host can be genetically manipulated. A pilot screen of 1,266 compounds with known pharmaceutical activities identified 15 (~1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Compounds identified through this screen exhibited anti-fungal activity in mice. We recently expanded this system and devised a C. elegans-C. neoformans assay that can be performed in liquid media. The SPECIFIC AIMS are as follows: Aim 1. Automate and expand the C. elegans-C. albicans assay. Aim 2. Standardize, automate and expand the C. elegans-C. neoformans assay. Aim 3. Validate the C. albicans and C. neoformans assays: a. Confirm antifungal activity, b. Develop a quantitative read-out of drug activity, c. Evaluate the MIC of "hit" compounds against a variety of fungi, d. Evaluate the toxicity of compounds against mammalian cells, e. Prioritize compounds, and, f. Evaluate the role of selected compounds in C. elegans immune response. In vivo evaluation of libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery, such as finding new classes of compounds and solving the bottleneck of toxicity/efficacy testing. In addition to the compounds that have direct antifungal activity, the C. elegans- based assays may help identify compounds that affect virulence factors or immuno-modulate evolutionary preserved elements of the host response to fungi. Moreover, developing these two antifungal assays will enable us in the future to perform comparative analyses between compounds identified in C. albicans and C. neoformans screens and to identify compounds that have broad antifungal activity. PUBLIC HEALTH RELEVANCE: There is an urgent need for the development of new antifungal agents to combat the increasing number of fungal infections and the development of antifungal resistance. The main objective of this application is the development of automated, high throughput, whole animal Caenorhabditis elegans-based assays that can be used to screen chemical compounds and identify those with antifungal activity. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery, such as finding new classes of compounds and solving the bottleneck of toxicity/efficacy testing.